Over-expression of Bovine (Bos taurus) BTN1A1 Gene Promotes Lipid Droplet Synthesis in Mammary Epithelial Cells
ZHANG Meng-Lu*, FAN Xiao-Na*, XING Zhi-Yang, WANG Yue-Ying, WANG Jiang, CHU Bei-Bei, HAN Li-Qiang**, YANG Guo-Yu**
College of Animal Science and Veterinary Medicine/Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture, Henan Agricultural University, Zhengzhou 450002, China
Abstract As a lipid droplet protein, butyrophilin subfamily 1 member A1 (BTN1A1) is associated with the formation and secretion of milk fat globules.In order to investigate the effect of over-expression bovine (Bos taurus) BTN1A1 on lipid droplets in mammary epithelial cells, the coding region of BTN1A1 gene was amplified by PCR, and Lenti-BTN1A1 lentivirus recombinant vector was constructed.The virus was packaged into recombinant vector and infected bovine mammary epithelial cells.BTN1A1 over-expression cell lines were selected by applying puromycin in cell culture medium.DNA was extracted from the over-expressed cell lines and genotypes were identified.Expression of BTN1A1 gene and protein was detected by qRT-PCR and Western blot.The effects of BTN1A1 over-expression on cell lipid droplets were analyzed by fluorescent labeling with Nile red.BTN1A1-pEGFP-N1 vector was constructed to observe the co-localization of BTN1A1 protein and lipid droplet.The results showed that the CDS sequence of the cloned BTN1A1 gene was about 1 580 bp.The lentiviral vector recombinant with BTN1A1 was constructed.BTN1A1 cell line was selecteded by 5 μg/mL purinomycin.Genotypic identification showed that exogenous BTN1A1 gene was successfully inserted into the cell genome.Compared with control group, the expression level of BTN1A1 gene mRNA was 489 times greater (P<0.001) ,while protein expression was also increased in over-expression BTN1A1 cell line.Fluorescence values of lipid droplets showed that over-expression BTN1A1 significantly increased lipid droplet content with the extension of cell culture time (P<0.01).BTN1A1 protein was found to co-localized with lipid droplets in cells.In this study, over-expression BTN1A1 promoted lipid droplet synthesis in mammary epithelial cells.This study provides basic data to elucidate the mechanism of lipid droplet synthesis in mammary epithalial cells of dairy cows.
[1] 杨浩, 李新建, 程佳, 等.2012.慢病毒介导shRNA干扰颗粒蛋白前体(GRN)促进猪前体脂肪细胞分化[J].农业生物技术学报, 20(6): 599-605. (Yang H, LI X J, Cheng J, et al.2012.Lentivirus mediated granulin shRNA interference silencing promotes pig preadipocyte differentiation[J].Journal of Agricultural Biotechnology, 20(6): 599-605.) [2] 张倡珲, 郝娟, 罗军, 等.2017.干扰TIP47 基因对奶山羊乳腺上皮细胞脂滴形成及相关基因表达的影响[J].农业生物技术学报, 25(2): 250-257. (Zhang C H, Hao J, Luo J, et al.2017.Effects of RNAi expression of TIP47 on lipid droplet formation and the expression of related genes in goat (Capra hircus) mammary epithelial cells[J].Journal of Agricultural Biotechnology, 25(2): 250-257.) [3] Bionaz M, Loor J J.2008.Gene networks driving bovine milk fat synthesis during the lactation cycle[J].Bmc Genomics, 9(1): 366. [4] Brasaemle D L.2007.The perilipin family of structural lipid droplet proteins: Stabilization of lipid droplets and control of lipolysis[J].Journal of Lipid Research, 48(12): 2547-2559. [5] Cohen B C, Shamay A, Argov-argaman N.2015.Regulation of lipid droplet size in mammary epithelial cells by remodeling of membrane lipid composition-a potential mechanism[J].PLos One, 10(3): e0121645. [6] Franke W W, Heid H W, Grund C, et al.1981.Antibodies to the major insoluble milk fat globule membrane-associated protein: Specific location in apical regions of lactating epithelial cells[J].The Journal of Cell Biology, 89(3): 485-494. [7] Holzmueller W, Kulozik U.2016.Isolation of milk fat globule membrane (MFGM) material by coagulation and diafiltration of buttermilk[J].International Dairy Journal, 63: 88-91. [8] Jeong J, Rao A U, Xu J, et al.2009.The PRY/SPRY/B30.2 domain of butyrophilin 1A1 (BTN1A1) binds to xanthine oxidoreductase: Implications for the function of BTN1A1 in the mammary gland and other tissues[J].Journal of Biological Chemistry, 284(33): 22444-22456. [9] Jeong J, Lisinski I, Kadegowda A K G, et al.2013.A test of current models for the mechanism of milk-lipid droplet secretion[J].Traffic, 14(9): 974-986. [10] Lovegrove J A, Givens D I.2016.Dairy food products: Good or bad for cardiometabolic disease?[J].Nutrition Research Reviews, 29(2): 249-267. [11] Lucey J A, Otter D, Horne D S, et al.2017.A 100-Year Review: Progress on the chemistry of milk and its components[J].Journal of Dairy Science, 100(12): 9916-9932. [12] Mcmanaman J L, Palmer C A, Anderson S, et al.2004.Regulation of milk lipid formation and secretion in the mouse mammary gland[J].Advances in Experimental Medicine & Biology, 554: 263-279. [13] Moriya H, Uchida K, Okajima T, et al.2011.Secretion of three enzymes for fatty acid synthesis into mouse milk in association with fat globules, and rapid decrease of the secreted enzymes by treatment with rapamycin[J].Archives of Biochemistry and Biophysics, 508(1): 87-92. [14] Nguyen H T H, Ong L, Hoque A, et al.2017.A proteomic characterization shows differences in the milk fat globule membrane of buffalo and bovine milk[J].Food Bioscience, 19: 7-16. [15] Ogg S L, Weldon A K, Dobbie L, et al.2004.Expression of butyrophilin (btn1a1) in lactating mammary gland is essential for the regulated secretion of milk-lipid droplets[J].Proceedings of the National Academy of Sciences of the USA, 101(27): 10084-10089. [16] Pyc M, Cai, Y Q, Greer, M S, et al.2017.Turning over a new leaf in lipid droplet biology[J].Trends in Plant Science, 22(7): 596-609. [17] Reinhardt T A, Lippolis J D.2008.Developmental changes in the milk fat globule membrane proteome during the transition from colostrum to milk[J].Journal of Dairy Science, 91(6): 2307-2318. [18] Robenek H, Hofnagel O, Buers I, et al.2006.Butyrophilin controls milk fat globule secretion[J].Proceedings of the National Academy Sciences of the USA, 103(27): 10385-10390. [19] Shi H B, Yu K, Luo J, et al.2015.Adipocyte differentiation-related protein promotes lipid accumulation in goat mammary epithelial cells[J].Journal of Dairy Science, 98(10): 6954-6964. [20] Tauchisato K, Ozeki S, Houjou T, et al.2002.The surface of lipid droplets is a phospholipid monolayer with a unique fatty acid composition[J].Journal of Biological Chemistry, 277(46): 44507-44512. [21] Wilfling F, Haas J T, Walther T C, et al.2014.Lipid droplet biogenesis[J].Current Opinion in Cell Biology, 29: 39-45. [22] Wu C C, Howell K E, Neville M C, et al.2000.Proteomics reveal a link between the endoplasmic reticulum and lipid secretory mechanisms in mammary epithelial cells[J].Electrophoresis, 21(16): 3470-3482.
